CN108285900A - A kind of recombination algin catenase and its construction method and application - Google Patents
A kind of recombination algin catenase and its construction method and application Download PDFInfo
- Publication number
- CN108285900A CN108285900A CN201810325487.9A CN201810325487A CN108285900A CN 108285900 A CN108285900 A CN 108285900A CN 201810325487 A CN201810325487 A CN 201810325487A CN 108285900 A CN108285900 A CN 108285900A
- Authority
- CN
- China
- Prior art keywords
- algin catenase
- algin
- aly
- cob
- expression vector
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 235000010443 alginic acid Nutrition 0.000 title claims abstract description 78
- 229920000615 alginic acid Polymers 0.000 title claims abstract description 78
- 230000006798 recombination Effects 0.000 title abstract description 17
- 238000005215 recombination Methods 0.000 title abstract description 17
- 238000010276 construction Methods 0.000 title abstract description 5
- 239000013604 expression vector Substances 0.000 claims abstract description 22
- 230000014509 gene expression Effects 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 8
- 230000015556 catabolic process Effects 0.000 claims abstract description 7
- 238000006731 degradation reaction Methods 0.000 claims abstract description 7
- 238000000855 fermentation Methods 0.000 claims abstract description 6
- 230000004151 fermentation Effects 0.000 claims abstract description 6
- 238000010353 genetic engineering Methods 0.000 claims abstract description 6
- 235000013305 food Nutrition 0.000 claims abstract description 3
- 108090000790 Enzymes Proteins 0.000 claims description 47
- 102000004190 Enzymes Human genes 0.000 claims description 46
- 241000894006 Bacteria Species 0.000 claims description 26
- 108090000623 proteins and genes Proteins 0.000 claims description 19
- 241000588724 Escherichia coli Species 0.000 claims description 17
- 210000004027 cell Anatomy 0.000 claims description 16
- 150000001413 amino acids Chemical group 0.000 claims description 13
- 239000002773 nucleotide Substances 0.000 claims description 9
- 125000003729 nucleotide group Chemical group 0.000 claims description 9
- 239000013612 plasmid Substances 0.000 claims description 8
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 7
- 229940072056 alginate Drugs 0.000 claims description 7
- 108010004131 poly(beta-D-mannuronate) lyase Proteins 0.000 claims description 7
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 claims description 6
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 244000063299 Bacillus subtilis Species 0.000 claims description 4
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 4
- 241000238631 Hexapoda Species 0.000 claims description 4
- 238000007792 addition Methods 0.000 claims description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 4
- 241000187747 Streptomyces Species 0.000 claims description 3
- 239000003292 glue Substances 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- 241000186046 Actinomyces Species 0.000 claims description 2
- 241000228245 Aspergillus niger Species 0.000 claims description 2
- 241000255789 Bombyx mori Species 0.000 claims description 2
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 claims description 2
- 241000233866 Fungi Species 0.000 claims description 2
- 241001138401 Kluyveromyces lactis Species 0.000 claims description 2
- 241000235058 Komagataella pastoris Species 0.000 claims description 2
- 241000499912 Trichoderma reesei Species 0.000 claims description 2
- 241000223261 Trichoderma viride Species 0.000 claims description 2
- -1 alginate oligosaccharides Chemical class 0.000 claims description 2
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims description 2
- 230000000593 degrading effect Effects 0.000 claims description 2
- 230000002538 fungal effect Effects 0.000 claims description 2
- 210000003292 kidney cell Anatomy 0.000 claims description 2
- 235000014655 lactic acid Nutrition 0.000 claims description 2
- 239000004310 lactic acid Substances 0.000 claims description 2
- 229920001542 oligosaccharide Polymers 0.000 claims description 2
- 210000001938 protoplast Anatomy 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
- 238000006467 substitution reaction Methods 0.000 claims description 2
- 239000013598 vector Substances 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 4
- 108091028043 Nucleic acid sequence Proteins 0.000 claims 3
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims 2
- KVZLHPXEUGJPAH-UHFFFAOYSA-N 2-oxidanylpropanoic acid Chemical compound CC(O)C(O)=O.CC(O)C(O)=O KVZLHPXEUGJPAH-UHFFFAOYSA-N 0.000 claims 1
- WFJIVOKAWHGMBH-UHFFFAOYSA-N 4-hexylbenzene-1,3-diol Chemical compound CCCCCCC1=CC=C(O)C=C1O WFJIVOKAWHGMBH-UHFFFAOYSA-N 0.000 claims 1
- 241000228212 Aspergillus Species 0.000 claims 1
- 241000699800 Cricetinae Species 0.000 claims 1
- 241000588722 Escherichia Species 0.000 claims 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims 1
- 241000124008 Mammalia Species 0.000 claims 1
- 241000206572 Rhodophyta Species 0.000 claims 1
- 210000002421 cell wall Anatomy 0.000 claims 1
- 230000001413 cellular effect Effects 0.000 claims 1
- 238000012217 deletion Methods 0.000 claims 1
- 230000037430 deletion Effects 0.000 claims 1
- 239000004615 ingredient Substances 0.000 claims 1
- 229960000448 lactic acid Drugs 0.000 claims 1
- 229910052708 sodium Inorganic materials 0.000 claims 1
- 230000002195 synergetic effect Effects 0.000 claims 1
- 230000009261 transgenic effect Effects 0.000 claims 1
- 241000512259 Ascophyllum nodosum Species 0.000 abstract description 7
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 abstract description 4
- 238000012545 processing Methods 0.000 abstract description 4
- 235000010413 sodium alginate Nutrition 0.000 abstract description 4
- 229940005550 sodium alginate Drugs 0.000 abstract description 4
- 239000000661 sodium alginate Substances 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 3
- 241001474374 Blennius Species 0.000 abstract description 2
- 230000001580 bacterial effect Effects 0.000 abstract description 2
- 230000007613 environmental effect Effects 0.000 abstract description 2
- 239000000203 mixture Substances 0.000 abstract description 2
- 239000002699 waste material Substances 0.000 abstract description 2
- 238000012215 gene cloning Methods 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 43
- 230000000694 effects Effects 0.000 description 18
- 108020004414 DNA Proteins 0.000 description 11
- 239000007788 liquid Substances 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Inorganic materials [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 9
- 239000006228 supernatant Substances 0.000 description 8
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- 235000000346 sugar Nutrition 0.000 description 6
- 102000018120 Recombinases Human genes 0.000 description 5
- 108010091086 Recombinases Proteins 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 229920000985 (beta-D-Mannuronate)n Polymers 0.000 description 4
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 150000004676 glycans Chemical class 0.000 description 4
- 102000057593 human F8 Human genes 0.000 description 4
- 229920001282 polysaccharide Polymers 0.000 description 4
- 239000005017 polysaccharide Substances 0.000 description 4
- 229940047431 recombinate Drugs 0.000 description 4
- 238000012795 verification Methods 0.000 description 4
- 108090000856 Lyases Proteins 0.000 description 3
- 102000004317 Lyases Human genes 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 229910021529 ammonia Inorganic materials 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000013613 expression plasmid Substances 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 241000607284 Vibrio sp. Species 0.000 description 2
- 108010093581 aspartyl-proline Proteins 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 108010089804 glycyl-threonine Proteins 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 1
- 102100021324 5-azacytidine-induced protein 2 Human genes 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- WDIYWDJLXOCGRW-ACZMJKKPSA-N Ala-Asp-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WDIYWDJLXOCGRW-ACZMJKKPSA-N 0.000 description 1
- FVSOUJZKYWEFOB-KBIXCLLPSA-N Ala-Gln-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)N FVSOUJZKYWEFOB-KBIXCLLPSA-N 0.000 description 1
- NBTGEURICRTMGL-WHFBIAKZSA-N Ala-Gly-Ser Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O NBTGEURICRTMGL-WHFBIAKZSA-N 0.000 description 1
- MDNAVFBZPROEHO-UHFFFAOYSA-N Ala-Lys-Val Natural products CC(C)C(C(O)=O)NC(=O)C(NC(=O)C(C)N)CCCCN MDNAVFBZPROEHO-UHFFFAOYSA-N 0.000 description 1
- CYBJZLQSUJEMAS-LFSVMHDDSA-N Ala-Phe-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](C)N)O CYBJZLQSUJEMAS-LFSVMHDDSA-N 0.000 description 1
- SSQHYGLFYWZWDV-UVBJJODRSA-N Ala-Val-Trp Chemical compound CC(C)[C@H](NC(=O)[C@H](C)N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(O)=O SSQHYGLFYWZWDV-UVBJJODRSA-N 0.000 description 1
- 241001148110 Alteromonas sp. M1 Species 0.000 description 1
- AUZAXCPWMDBWEE-HJGDQZAQSA-N Arg-Thr-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O AUZAXCPWMDBWEE-HJGDQZAQSA-N 0.000 description 1
- ULBHWNVWSCJLCO-NHCYSSNCSA-N Arg-Val-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCN=C(N)N ULBHWNVWSCJLCO-NHCYSSNCSA-N 0.000 description 1
- RZVVKNIACROXRM-ZLUOBGJFSA-N Asn-Ala-Asp Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CC(=O)N)N RZVVKNIACROXRM-ZLUOBGJFSA-N 0.000 description 1
- BRCVLJZIIFBSPF-ZLUOBGJFSA-N Asn-Ala-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)N)N BRCVLJZIIFBSPF-ZLUOBGJFSA-N 0.000 description 1
- XVVOVPFMILMHPX-ZLUOBGJFSA-N Asn-Asp-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O XVVOVPFMILMHPX-ZLUOBGJFSA-N 0.000 description 1
- PNHQRQTVBRDIEF-CIUDSAMLSA-N Asn-Leu-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(=O)N)N PNHQRQTVBRDIEF-CIUDSAMLSA-N 0.000 description 1
- CDGHMJJJHYKMPA-DLOVCJGASA-N Asn-Phe-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CC(=O)N)N CDGHMJJJHYKMPA-DLOVCJGASA-N 0.000 description 1
- KDFQZBWWPYQBEN-ZLUOBGJFSA-N Asp-Ala-Asn Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)O)N KDFQZBWWPYQBEN-ZLUOBGJFSA-N 0.000 description 1
- SDHFVYLZFBDSQT-DCAQKATOSA-N Asp-Arg-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(=O)O)N SDHFVYLZFBDSQT-DCAQKATOSA-N 0.000 description 1
- RDRMWJBLOSRRAW-BYULHYEWSA-N Asp-Asn-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O RDRMWJBLOSRRAW-BYULHYEWSA-N 0.000 description 1
- VPSHHQXIWLGVDD-ZLUOBGJFSA-N Asp-Asp-Asp Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O VPSHHQXIWLGVDD-ZLUOBGJFSA-N 0.000 description 1
- VILLWIDTHYPSLC-PEFMBERDSA-N Asp-Glu-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VILLWIDTHYPSLC-PEFMBERDSA-N 0.000 description 1
- OVPHVTCDVYYTHN-AVGNSLFASA-N Asp-Glu-Phe Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 OVPHVTCDVYYTHN-AVGNSLFASA-N 0.000 description 1
- JUWZKMBALYLZCK-WHFBIAKZSA-N Asp-Gly-Asn Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O JUWZKMBALYLZCK-WHFBIAKZSA-N 0.000 description 1
- ZSVJVIOVABDTTL-YUMQZZPRSA-N Asp-Gly-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC(=O)O)N ZSVJVIOVABDTTL-YUMQZZPRSA-N 0.000 description 1
- LDLZOAJRXXBVGF-GMOBBJLQSA-N Asp-Ile-Met Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CC(=O)O)N LDLZOAJRXXBVGF-GMOBBJLQSA-N 0.000 description 1
- PAYPSKIBMDHZPI-CIUDSAMLSA-N Asp-Leu-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O PAYPSKIBMDHZPI-CIUDSAMLSA-N 0.000 description 1
- DONWIPDSZZJHHK-HJGDQZAQSA-N Asp-Lys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(=O)O)N)O DONWIPDSZZJHHK-HJGDQZAQSA-N 0.000 description 1
- PLNJUJGNLDSFOP-UWJYBYFXSA-N Asp-Tyr-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O PLNJUJGNLDSFOP-UWJYBYFXSA-N 0.000 description 1
- 241000351920 Aspergillus nidulans Species 0.000 description 1
- 241000589149 Azotobacter vinelandii Species 0.000 description 1
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 241001135695 Cobetia marina Species 0.000 description 1
- 241000086967 Cobetia sp. Species 0.000 description 1
- 241001459439 Corynebacterium sp. ALY-1 Species 0.000 description 1
- NQSUTVRXXBGVDQ-LKXGYXEUSA-N Cys-Asn-Thr Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O NQSUTVRXXBGVDQ-LKXGYXEUSA-N 0.000 description 1
- 201000003883 Cystic fibrosis Diseases 0.000 description 1
- AEMOLEFTQBMNLQ-BZINKQHNSA-N D-Guluronic Acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@@H](O)[C@H]1O AEMOLEFTQBMNLQ-BZINKQHNSA-N 0.000 description 1
- 241000588697 Enterobacter cloacae Species 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- 241000672609 Escherichia coli BL21 Species 0.000 description 1
- 241000589565 Flavobacterium Species 0.000 description 1
- 241000589564 Flavobacterium sp. Species 0.000 description 1
- BVELAHPZLYLZDJ-HGNGGELXSA-N Gln-His-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(O)=O BVELAHPZLYLZDJ-HGNGGELXSA-N 0.000 description 1
- WOSRKEJQESVHGA-CIUDSAMLSA-N Glu-Arg-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O WOSRKEJQESVHGA-CIUDSAMLSA-N 0.000 description 1
- CKOFNWCLWRYUHK-XHNCKOQMSA-N Glu-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)O)N)C(=O)O CKOFNWCLWRYUHK-XHNCKOQMSA-N 0.000 description 1
- HPJLZFTUUJKWAJ-JHEQGTHGSA-N Glu-Gly-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O HPJLZFTUUJKWAJ-JHEQGTHGSA-N 0.000 description 1
- SWRVAQHFBRZVNX-GUBZILKMSA-N Glu-Lys-Asn Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O SWRVAQHFBRZVNX-GUBZILKMSA-N 0.000 description 1
- SYWCGQOIIARSIX-SRVKXCTJSA-N Glu-Pro-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O SYWCGQOIIARSIX-SRVKXCTJSA-N 0.000 description 1
- BDISFWMLMNBTGP-NUMRIWBASA-N Glu-Thr-Asp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O BDISFWMLMNBTGP-NUMRIWBASA-N 0.000 description 1
- CQGBSALYGOXQPE-HTUGSXCWSA-N Glu-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)O CQGBSALYGOXQPE-HTUGSXCWSA-N 0.000 description 1
- AIJAPFVDBFYNKN-WHFBIAKZSA-N Gly-Asn-Asp Chemical compound C([C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)CN)C(=O)N AIJAPFVDBFYNKN-WHFBIAKZSA-N 0.000 description 1
- JPWIMMUNWUKOAD-STQMWFEESA-N Gly-Asp-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)CN JPWIMMUNWUKOAD-STQMWFEESA-N 0.000 description 1
- NTOWAXLMQFKJPT-YUMQZZPRSA-N Gly-Glu-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)CN NTOWAXLMQFKJPT-YUMQZZPRSA-N 0.000 description 1
- INLIXXRWNUKVCF-JTQLQIEISA-N Gly-Gly-Tyr Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 INLIXXRWNUKVCF-JTQLQIEISA-N 0.000 description 1
- SWQALSGKVLYKDT-ZKWXMUAHSA-N Gly-Ile-Ala Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O SWQALSGKVLYKDT-ZKWXMUAHSA-N 0.000 description 1
- SWQALSGKVLYKDT-UHFFFAOYSA-N Gly-Ile-Ala Natural products NCC(=O)NC(C(C)CC)C(=O)NC(C)C(O)=O SWQALSGKVLYKDT-UHFFFAOYSA-N 0.000 description 1
- LOEANKRDMMVOGZ-YUMQZZPRSA-N Gly-Lys-Asp Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)N[C@@H](CC(O)=O)C(O)=O LOEANKRDMMVOGZ-YUMQZZPRSA-N 0.000 description 1
- GMTXWRIDLGTVFC-IUCAKERBSA-N Gly-Lys-Glu Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O GMTXWRIDLGTVFC-IUCAKERBSA-N 0.000 description 1
- OQQKUTVULYLCDG-ONGXEEELSA-N Gly-Lys-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCCCN)NC(=O)CN)C(O)=O OQQKUTVULYLCDG-ONGXEEELSA-N 0.000 description 1
- IGOYNRWLWHWAQO-JTQLQIEISA-N Gly-Phe-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 IGOYNRWLWHWAQO-JTQLQIEISA-N 0.000 description 1
- ZLCLYFGMKFCDCN-XPUUQOCRSA-N Gly-Ser-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CO)NC(=O)CN)C(O)=O ZLCLYFGMKFCDCN-XPUUQOCRSA-N 0.000 description 1
- UIQGJYUEQDOODF-KWQFWETISA-N Gly-Tyr-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 UIQGJYUEQDOODF-KWQFWETISA-N 0.000 description 1
- COZMNNJEGNPDED-HOCLYGCPSA-N Gly-Val-Trp Chemical compound [H]NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O COZMNNJEGNPDED-HOCLYGCPSA-N 0.000 description 1
- VBOFRJNDIOPNDO-YUMQZZPRSA-N His-Gly-Asn Chemical compound C1=C(NC=N1)C[C@@H](C(=O)NCC(=O)N[C@@H](CC(=O)N)C(=O)O)N VBOFRJNDIOPNDO-YUMQZZPRSA-N 0.000 description 1
- LVWIJITYHRZHBO-IXOXFDKPSA-N His-Leu-Thr Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LVWIJITYHRZHBO-IXOXFDKPSA-N 0.000 description 1
- WYSJPCTWSBJFCO-AVGNSLFASA-N His-Met-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CC1=CN=CN1)N WYSJPCTWSBJFCO-AVGNSLFASA-N 0.000 description 1
- KFQDSSNYWKZFOO-LSJOCFKGSA-N His-Val-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O KFQDSSNYWKZFOO-LSJOCFKGSA-N 0.000 description 1
- JLWLMGADIQFKRD-QSFUFRPTSA-N Ile-His-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CN=CN1 JLWLMGADIQFKRD-QSFUFRPTSA-N 0.000 description 1
- 108090001007 Interleukin-8 Proteins 0.000 description 1
- 241000256602 Isoptera Species 0.000 description 1
- 241000588748 Klebsiella Species 0.000 description 1
- 201000008225 Klebsiella pneumonia Diseases 0.000 description 1
- 241000588747 Klebsiella pneumoniae Species 0.000 description 1
- KWTVLKBOQATPHJ-SRVKXCTJSA-N Leu-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(C)C)N KWTVLKBOQATPHJ-SRVKXCTJSA-N 0.000 description 1
- BQSLGJHIAGOZCD-CIUDSAMLSA-N Leu-Ala-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O BQSLGJHIAGOZCD-CIUDSAMLSA-N 0.000 description 1
- GRZSCTXVCDUIPO-SRVKXCTJSA-N Leu-Arg-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(O)=O GRZSCTXVCDUIPO-SRVKXCTJSA-N 0.000 description 1
- KSZCCRIGNVSHFH-UWVGGRQHSA-N Leu-Arg-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O KSZCCRIGNVSHFH-UWVGGRQHSA-N 0.000 description 1
- GPICTNQYKHHHTH-GUBZILKMSA-N Leu-Gln-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O GPICTNQYKHHHTH-GUBZILKMSA-N 0.000 description 1
- LAPSXOAUPNOINL-YUMQZZPRSA-N Leu-Gly-Asp Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC(O)=O LAPSXOAUPNOINL-YUMQZZPRSA-N 0.000 description 1
- BJWKOATWNQJPSK-SRVKXCTJSA-N Leu-Met-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N BJWKOATWNQJPSK-SRVKXCTJSA-N 0.000 description 1
- WMIOEVKKYIMVKI-DCAQKATOSA-N Leu-Pro-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O WMIOEVKKYIMVKI-DCAQKATOSA-N 0.000 description 1
- BMVFXOQHDQZAQU-DCAQKATOSA-N Leu-Pro-Asp Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(=O)O)C(=O)O)N BMVFXOQHDQZAQU-DCAQKATOSA-N 0.000 description 1
- IRMLZWSRWSGTOP-CIUDSAMLSA-N Leu-Ser-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O IRMLZWSRWSGTOP-CIUDSAMLSA-N 0.000 description 1
- QWWPYKKLXWOITQ-VOAKCMCISA-N Leu-Thr-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(C)C QWWPYKKLXWOITQ-VOAKCMCISA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- MPOHDJKRBLVGCT-CIUDSAMLSA-N Lys-Ala-Asn Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCCCN)N MPOHDJKRBLVGCT-CIUDSAMLSA-N 0.000 description 1
- UWKNTTJNVSYXPC-CIUDSAMLSA-N Lys-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN UWKNTTJNVSYXPC-CIUDSAMLSA-N 0.000 description 1
- SWWCDAGDQHTKIE-RHYQMDGZSA-N Lys-Arg-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SWWCDAGDQHTKIE-RHYQMDGZSA-N 0.000 description 1
- KEPWSUPUFAPBRF-DKIMLUQUSA-N Lys-Ile-Phe Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O KEPWSUPUFAPBRF-DKIMLUQUSA-N 0.000 description 1
- YXPJCVNIDDKGOE-MELADBBJSA-N Lys-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)N)C(=O)O YXPJCVNIDDKGOE-MELADBBJSA-N 0.000 description 1
- VWPJQIHBBOJWDN-DCAQKATOSA-N Lys-Val-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O VWPJQIHBBOJWDN-DCAQKATOSA-N 0.000 description 1
- QLFAPXUXEBAWEK-NHCYSSNCSA-N Lys-Val-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O QLFAPXUXEBAWEK-NHCYSSNCSA-N 0.000 description 1
- IKXQOBUBZSOWDY-AVGNSLFASA-N Lys-Val-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](CCCCN)N IKXQOBUBZSOWDY-AVGNSLFASA-N 0.000 description 1
- UZVWDRPUTHXQAM-FXQIFTODSA-N Met-Asp-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O UZVWDRPUTHXQAM-FXQIFTODSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 1
- QCHNRQQVLJYDSI-DLOVCJGASA-N Phe-Asn-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 QCHNRQQVLJYDSI-DLOVCJGASA-N 0.000 description 1
- MGECUMGTSHYHEJ-QEWYBTABSA-N Phe-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 MGECUMGTSHYHEJ-QEWYBTABSA-N 0.000 description 1
- PTDAGKJHZBGDKD-OEAJRASXSA-N Phe-Thr-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N)O PTDAGKJHZBGDKD-OEAJRASXSA-N 0.000 description 1
- ZYNBEWGJFXTBDU-ACRUOGEOSA-N Phe-Tyr-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CC2=CC=CC=C2)N ZYNBEWGJFXTBDU-ACRUOGEOSA-N 0.000 description 1
- MWQXFDIQXIXPMS-UNQGMJICSA-N Phe-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC1=CC=CC=C1)N)O MWQXFDIQXIXPMS-UNQGMJICSA-N 0.000 description 1
- 206010035717 Pneumonia klebsiella Diseases 0.000 description 1
- MTHRMUXESFIAMS-DCAQKATOSA-N Pro-Asn-Lys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O MTHRMUXESFIAMS-DCAQKATOSA-N 0.000 description 1
- SXMSEHDMNIUTSP-DCAQKATOSA-N Pro-Lys-Asn Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O SXMSEHDMNIUTSP-DCAQKATOSA-N 0.000 description 1
- LEIKGVHQTKHOLM-IUCAKERBSA-N Pro-Pro-Gly Chemical compound OC(=O)CNC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 LEIKGVHQTKHOLM-IUCAKERBSA-N 0.000 description 1
- 241000519590 Pseudoalteromonas Species 0.000 description 1
- 241000589540 Pseudomonas fluorescens Species 0.000 description 1
- 241001391935 Pseudomonas sp. QD03 Species 0.000 description 1
- 241000589615 Pseudomonas syringae Species 0.000 description 1
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 1
- PZZJMBYSYAKYPK-UWJYBYFXSA-N Ser-Ala-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O PZZJMBYSYAKYPK-UWJYBYFXSA-N 0.000 description 1
- XVAUJOAYHWWNQF-ZLUOBGJFSA-N Ser-Asn-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O XVAUJOAYHWWNQF-ZLUOBGJFSA-N 0.000 description 1
- HEQPKICPPDOSIN-SRVKXCTJSA-N Ser-Asp-Tyr Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 HEQPKICPPDOSIN-SRVKXCTJSA-N 0.000 description 1
- IFPBAGJBHSNYPR-ZKWXMUAHSA-N Ser-Ile-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O IFPBAGJBHSNYPR-ZKWXMUAHSA-N 0.000 description 1
- HSWXBJCBYSWBPT-GUBZILKMSA-N Ser-Val-Val Chemical compound CC(C)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CO)C(C)C)C(O)=O HSWXBJCBYSWBPT-GUBZILKMSA-N 0.000 description 1
- 241000801167 Streptomyces sp. ALG5 Species 0.000 description 1
- 241001655322 Streptomycetales Species 0.000 description 1
- XDARBNMYXKUFOJ-GSSVUCPTSA-N Thr-Asp-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XDARBNMYXKUFOJ-GSSVUCPTSA-N 0.000 description 1
- VUSAEKOXGNEYNE-PBCZWWQYSA-N Thr-His-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)O VUSAEKOXGNEYNE-PBCZWWQYSA-N 0.000 description 1
- IGGFFPOIFHZYKC-PBCZWWQYSA-N Thr-His-Asp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC(=O)O)C(=O)O)N)O IGGFFPOIFHZYKC-PBCZWWQYSA-N 0.000 description 1
- ABWNZPOIUJMNKT-IXOXFDKPSA-N Thr-Phe-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O ABWNZPOIUJMNKT-IXOXFDKPSA-N 0.000 description 1
- NYQIZWROIMIQSL-VEVYYDQMSA-N Thr-Pro-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O NYQIZWROIMIQSL-VEVYYDQMSA-N 0.000 description 1
- MYNYCUXMIIWUNW-IEGACIPQSA-N Thr-Trp-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CCCCN)C(=O)O)N)O MYNYCUXMIIWUNW-IEGACIPQSA-N 0.000 description 1
- KZTLJLFVOIMRAQ-IHPCNDPISA-N Trp-Asn-Tyr Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O KZTLJLFVOIMRAQ-IHPCNDPISA-N 0.000 description 1
- ARKBYVBCEOWRNR-UBHSHLNASA-N Trp-Ser-Ser Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O ARKBYVBCEOWRNR-UBHSHLNASA-N 0.000 description 1
- WDIJBEWLXLQQKD-ULQDDVLXSA-N Tyr-Arg-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N)O WDIJBEWLXLQQKD-ULQDDVLXSA-N 0.000 description 1
- MBFJIHUHHCJBSN-AVGNSLFASA-N Tyr-Asn-Gln Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O MBFJIHUHHCJBSN-AVGNSLFASA-N 0.000 description 1
- ZOBLBMGJKVJVEV-BZSNNMDCSA-N Tyr-Lys-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)O)N)O ZOBLBMGJKVJVEV-BZSNNMDCSA-N 0.000 description 1
- PMHLLBKTDHQMCY-ULQDDVLXSA-N Tyr-Lys-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O PMHLLBKTDHQMCY-ULQDDVLXSA-N 0.000 description 1
- PSALWJCUIAQKFW-ACRUOGEOSA-N Tyr-Phe-Lys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N PSALWJCUIAQKFW-ACRUOGEOSA-N 0.000 description 1
- OQWNEUXPKHIEJO-NRPADANISA-N Val-Glu-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CO)C(=O)O)N OQWNEUXPKHIEJO-NRPADANISA-N 0.000 description 1
- BEGDZYNDCNEGJZ-XVKPBYJWSA-N Val-Gly-Gln Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O BEGDZYNDCNEGJZ-XVKPBYJWSA-N 0.000 description 1
- DIOSYUIWOQCXNR-ONGXEEELSA-N Val-Lys-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O DIOSYUIWOQCXNR-ONGXEEELSA-N 0.000 description 1
- HWNYVQMOLCYHEA-IHRRRGAJSA-N Val-Ser-Tyr Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N HWNYVQMOLCYHEA-IHRRRGAJSA-N 0.000 description 1
- 241000607598 Vibrio Species 0.000 description 1
- 206010047400 Vibrio infections Diseases 0.000 description 1
- 241001064682 Vibrio sp. O2 Species 0.000 description 1
- 241000594907 Vibrio sp. QY105 Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 1
- 108010005233 alanylglutamic acid Proteins 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- 102000004139 alpha-Amylases Human genes 0.000 description 1
- 108090000637 alpha-Amylases Proteins 0.000 description 1
- 229940024171 alpha-amylase Drugs 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 1
- 108010092854 aspartyllysine Proteins 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- AEMOLEFTQBMNLQ-UHFFFAOYSA-N beta-D-galactopyranuronic acid Natural products OC1OC(C(O)=O)C(O)C(O)C1O AEMOLEFTQBMNLQ-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- FSXRLASFHBWESK-UHFFFAOYSA-N dipeptide phenylalanyl-tyrosine Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FSXRLASFHBWESK-UHFFFAOYSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940119563 enterobacter cloacae Drugs 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 108010010096 glycyl-glycyl-tyrosine Proteins 0.000 description 1
- 108010077435 glycyl-phenylalanyl-glycine Proteins 0.000 description 1
- 108010085325 histidylproline Proteins 0.000 description 1
- 108010018006 histidylserine Proteins 0.000 description 1
- 230000015784 hyperosmotic salinity response Effects 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 108010044348 lysyl-glutamyl-aspartic acid Proteins 0.000 description 1
- 108010017391 lysylvaline Proteins 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 238000004886 process control Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 108010087846 prolyl-prolyl-glycine Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 150000003538 tetroses Chemical class 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 108010005834 tyrosyl-alanyl-glycine Proteins 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 108010027345 wheylin-1 peptide Proteins 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of recombination algin catenase and its construction method and applications, belong to biotechnology.Algin catenase Aly Cob according to the present invention are from environmental sample (at kelp stacking and mixture of plant produced waste material), the present invention utilizes the technical method of genetic engineering, it will be in the gene cloning to expression vector of the algin catenase, obtain can heterogenous expression algin catenase recombinant bacterial strain, the algin catenase Aly Cob that the strain fermentation generates have the function of that degradation sodium alginate prepares brown alga oligose.Algin catenase Aly Cob provided by the invention can be widely applied to agricultural, food, feed addition, medicine and seaweed processing and other fields.
Description
Technical field
The present invention relates to a kind of recombination algin catenase and its construction method and applications, belong to biotechnology.
Background technology
In recent years, flourishing with marine drug, the research of algal polysaccharides is paid more and more attention, and algin is it
One of.Algin is with a wide range of applications, and algin degradation product is due to antitumor, immunological regulation, hypoglycemic blood
The multiple biological activities such as fat have become brown alga acid product in the world and deeply research and develop the representative direction of high value added utilization and grind at present
Study carefully hot spot.Algin catenase digests production brown alga oligose and low-molecular-weight polysaccharide as toolenzyme has degradation condition mild,
The advantages that process control, yield is high, has gradually replaced traditional acid hydrolysis method and has become the major way of brown alga oligose production.This
Outside, this fermentoid also all has important researching value and application value in addition to that can prepare brown alga oligose in all various aspects, such as living
The preparation of property Tang oligosaccharide, the preparation of protoplast, the auxiliary treatment etc. of pulmonary cystic fibrosis.Therefore, algin catenase
Excavation hold out broad prospects.
It has been reported that algin catenase mostly be from kelp, with brown alga be eat marine animal and soil in microorganism
Middle acquisition, such as pseudoalteromonas (Pseudoaltermonas elyakovii), pseudomonad (Pseudomonas
Sp.QD03, Pseudomonas fluorescens, Pseudomonas syringae), nitrogen-fixing bacteria (Azotobacter
Vinelandii), vibrios (Vibrio sp.510-64, Vibrio sp.O2), Klebsiella (Klebsiella
Pneumonia), bacillus (Bacillus sp.ATB-1015), Flavobacterium (Flavobacterium sp.LXA), alternating
Monad (Alteromonas sp.M-1), bar bacterium (Corynebacterium sp.ALY-1), enterobacteria
(Enterobacter-Cloacae M-1), streptomycete (Streptomyces sp.ALG-5) etc..In addition, in a small number of virus
With algin catenase is had also discovered in bacteriophage.Most of microbes producing cellulase growth conditions is special, and yield of enzyme is low, and separation is tired
It is difficult.The gene for having had more than 20 kinds of algin catenases at present is cloned and is sequenced, and is constructed a variety of recombination algins and split
Enzyme gene engineering bacteria is solved, but recombination alginate lyase gene engineering expression is relatively low, limits the application of enzyme.Therefore, sharp
With genetic engineering means, further obtain high yield algin catenase engineering bacteria, by push algin catenase development and its
Application extension.
Invention content
The first purpose of the invention is to provide a kind of algin catenase Aly-Cob and its encoding genes.
Recombination algin catenase Aly-Cob provided by the present invention is obtained, ammonia by building Fosmid library screenings
Base sequence set has one of following feature：
1) 1-352s or 30-352 amino acids residue sequence of the SEQ ID NO.2 since aminoterminal in sequence table；
2) one or several amino acid substitutions, missing are carried out to amino acid sequence shown in SEQ ID NO.2 in sequence table
Or addition and formed have the active amino acid sequence of algin catenase.
The present invention also provides the encoding gene of algin catenase Aly-Cob (being named as aly-cob), have following cores
One of nucleotide sequence feature：
1) in sequence table SEQ ID NO.1 DNA (DNA) sequence；
2) in polynucleotide SEQ ID NO.2 amino acid sequences DNA (DNA) sequence；
3) one or several nucleotide are carried out to DNA (DNA) sequence of SEQ ID NO.1 in sequence table to take
Coding has the active nucleotide sequence of algin catenase obtained from generation, missing or addition.
The amino acid sequence and its nucleotide coding sequence of the recombination algin catenase of the present invention can also be according to predictions
Aly-Cob amino acid sequence and its artificial synthesized acquisition of nucleotide coding sequence.
Second object of the present invention is to provide the recombinant expression plasmid containing the alginate lyase gene and again
Group engineering strain.
The recombinant expression plasmid is the expression vector for carrying the gene for encoding the inscribe algin catenase, is
Refer to coli expression carrier, Yeast expression carrier, hay bacillus expression vector, Corynebacterium glutamicum expression vector, lactic acid bacteria
Expression vector, streptomyces expression vector, phage vector, filamentous fungi expression vector, plant expression vector, insect expression carry
Body or mammalian cell expression vector.The coli expression carrier, can be pET-21a (+), pET-28a (+),
pET-32a(+).The hay bacillus expression vector can be pMA5.The Yeast expression carrier can be pPIC3.5k.It is described
Corynebacterium glutamicum expression vector can be pDXW-10.
The recombination engineered strain is recombinant bacterium or transgenosis for recombinantly expressing inscribe algin catenase
Cell line, refer to e. coli host cell (such as Escherichia coli BL21, Escherichia coli JM109,
Escherichia coli DH5 α etc.), yeast host cells (such as Saccharomyces cerevisiae, Pichiapas
Toris, Kluyveromyces lactis etc.), hay bacillus host cell (such as Bacillus subtilis R25,
Bacillus subtilis 9920 etc.), corynebacterium glutamicum (Corynebacterium glutamicum etc.), actinomyces
Host cell (such as Streptomyces spp.), filamentous fungal host cell (such as Trichoderma viride,
Trichoderma reesei, Aspergillus niger, Aspergillus nidulans etc.), insect cell (such as
Bombyx mori, Antharaea eucalypti etc.), mammalian cell (such as Chinese hamster ovary cell CHO, immature storehouse
Mouse kidney cell BHK, CHL cells CHL etc.) in one kind.
Specifically, the recombination engineered strain can be E.coli BL21pLysS/pET-28a (+)-aly-cob.
Third object of the present invention is to provide a kind of methods of Prepare restructuring algin catenase Aly-Cob, are that will compile
Code gene aly-cob is cloned into expression vector, and imports host cell, to obtain recombination algin catenase.For example, will structure
In good recombinant bacterium E.coli BL21pLysS/pET-28a (+)-aly-cob access liquid LB, 37 DEG C of cultures to OD600Up to 0.8
Fermentation supernatant is collected, and collect thalline and be crushed, centrifugation is stayed after the IPTG inductions of final concentration of 0.5mmol/L are added in left and right
Merge with fermentation supernatant after taking supernatant, it is purified to obtain recombination algin catenase.
The present invention screens to obtain one section of alginate lyase gene sequence aly-cob using structure Fosmid library approach,
Gene code head of district 1059bp encodes 352 amino acid, wherein 1-29 amino acids encoded signal peptide, 30-352 ammonia
Base acid encoding algin catenase, molecular weight about 35kDa belong to 7 family of polysaceharide lyase.
The present invention Aly-Cob that heterogenous expression obtains in Escherichia coli, when using sodium alginate as substrate, in 45 DEG C, pH
There is highest enzymatic activity, not only degradable poly guluronic acid (PG) but also the polymannuronate (PM) that can degrade under conditions of 8.0,
But it is stronger to the specificity of the latter.Liquid matter analysis shows, the catabolite of sodium alginate is mainly two, three, tetrose, be on a small quantity five
Sugar and six sugar.The enzyme is in 1.5molL-1NaCl and 1molL-1KCl under the conditions of, enzyme activity, which is respectively increased, lives to protoenzyme
1.7 and 1.5 times.
Algin catenase Aly-Cob provided by the invention can individually or with other source algin degrading enzymes or other
Kind fermentoid is used in conjunction with, and is used to prepare brown alga oligose or is glued raw material progress viscosity reduction processing to the height containing alginate.
The algin catenase of the present invention can be widely used for chemical industry, agricultural, food and feed addition, medicine and seaweed heredity
The fields such as engineering.
Beneficial effects of the present invention：
Aly-Cob provided by the invention is a kind of recombination algin catenase of great potential, and advantage is mainly reflected in：
(1) the recombinant bacterium enzyme activity built is horizontal high, and the producing enzyme period is short；(2) foreign protein content is few in Aly-Cob crude enzyme liquids, is easily isolated
Purifying；(3) Aly-Cob has preferable thermal stability and the tolerance of pH and salt, has advantage in commercial Application；(4)
Aly-Cob can be used for efficiently preparing brown alga oligose and carry out the viscosity reduction processing of alginate-containing raw material.
Description of the drawings
Fig. 1：Alginate lyase gene aly-cob PCR amplifications
Fig. 2：Algin catenase recombinates the verification of bacteria plasmid double digestion
Fig. 3：Recombinate the catabolite liquid phase figure of algin catenase Aly-Cob
Fig. 4：Recombinate the catabolite mass spectrogram of algin catenase Aly-Cob
Specific implementation mode
Embodiment 1：Algin catenase Aly-Cob full-length genes are cloned and analysis
(1) it is directed to the mixture of environmental sample and production waste material at kelp stacking in kelp production processing factory, is passed through
Structure Fosmid Metagenomic library screenings obtain the gene of coding algin catenase.By the PCR amplification gene, design is drawn
5 ' ends when object in primer add restriction enzyme site BamH I and Hind III respectively, and primer sequence is：
AlyC-F：5’-CGGGATCCATGCGTAACACCCGTGTGC-3 ', what is marked with underscore is BamH I digestions position
Point；
AlyC-R：5’-CCCAAGCTTTCATTGCATCTCACCGCTCT-3 ', what is marked with underscore is Hind III enzymes
Enzyme site.
PCR conditions are：94 DEG C of pre-degenerations, 3min；94 DEG C, 30s；58 DEG C, 30s；72 DEG C, 1min；Totally 30 cycles, finally
72 DEG C, 10min.Agarose gel electrophoresis, which is shown in 1.0kb or so, a specific band (Fig. 1).It is coagulated from agarose
It cuts on glue, is purified using DNA gel QIAquick Gel Extraction Kit.
(2) DNA fragmentation of purifying is connected on cloning vector pMD19-T, conversion is thin to e. coli jm109 competence
It in born of the same parents, is incubated overnight on LB tablets (containing ampicillin), PCR verifications, selection is carried out after picking single bacterium colony Liquid Culture
There is the positive colony of band to be sequenced.
Sequencing result is analyzed in NCBI, as a result shows that the sequence is that algin catenase encoding gene (is named as
Aly-cob), gene code head of district 1059bp, nucleotide sequence is as shown in SEQ ID NO.1.The gene order is existed
Homologous comparison is carried out in ncbi database, finds the gene and 21022 (NZ_ of Cobetia marina strain JCM
CP017114 the algin catenase coded sequence similarity highest predicted in), reachable 90%, but gene NZ_CP017114
Exact function is simultaneously non-verified.
The algin catenase Aly-Cob of aly-cob codings is made of 352 amino acid, amino acid sequence such as SEQ
Shown in ID NO.2, the theoretical molecular weight of protein is about 35.7kDa.With SignalP (https：//www.plob.org/
Article/2404.html) the structural information of analysis algin catenase Aly-Cob, as a result shows that N-terminal starts to the 29th ammonia
Base acid is signal peptide sequence, and 30-352 amino acids belong to 7 family of polysaceharide lyase.By algin catenase of the present invention
The algin catenase AlgC-PL7 comparison discoveries of the amino acid sequence of Aly-Cob and the sources Cobetia sp.NAP1, the two phase
It is 92.33% like degree.But no matter the two has differences (embodiment 3) in enzyme activity or zymologic property.Thus speculate, this hair
The algin catenase of bright acquisition is a kind of new enzyme, has good research and application potential.
Embodiment 2：Recombinant bacterium is built and producing enzyme
The structure of recombinant bacterium E.coli BL21pLysS/pET-28a (+)-aly-cob：With restriction enzyme BamH I
Distinguish digestion objective gene sequence aly-cob and plasmid pET-28a (+) with Hind III, with T4 ligases be ligated and transformed into
In E.coli BL21pLysS.Picking single bacterium colony extracts recombinant plasmid after carrying out Liquid Culture, carries out PCR verifications to it, and make
Double digestion verification is carried out with BamH I and Hind III.Two segments that length is respectively 5.3kb and 1kb or so are obtained, are such as schemed
2, it was demonstrated that alginate lyase gene on successful clone to expression vector pET-28a (+) and is converted to expressive host
In E.coli BL21pLysS, this recombinant plasmid is named as pET-28a (+)-aly-cob, recombinant bacterium is named as E.coli
BL21pLysS/pET-28a(+)-aly-cob。
The induced expression of recombinant bacterium：By the recombinant bacterium built by 1% inoculum concentration access liquid LB (containing kanamycins and
Chloramphenicol) in, 37 DEG C of cultures to OD600Up to 0.8 or so, the IPTG that final concentration of 0.5mmol/L is added is induced, fermentation knot
Centrifuging and taking supernatant after beam；Thalline is resuspended after being cleaned with PBS, is centrifuged after ultrasonication and leaves and takes supernatant；Hair is measured respectively using DNS methods
Algin catenase vigor in ferment supernatant and broken supernatant, the two adduction are algin catenase enzyme activity.The results show that negative
Enzyme activity, the enzyme activity of recombinant bacterium E.coli BL21pLysS/pET-28a (+)-aly-cob is not detected in control (unloaded recombinant bacterium)
For 466U/mL.1 enzyme activity unit (U) is defined as：The 1mL enzyme solutions enzyme amount per minute generated needed for 1 μ g reduced sugars.
Objective gene sequence aly-cob is expressed using other plasmids or host, derivational expression method referring to
E.coli BL21pLysS/pET-28a (+)-aly-cob, measures the enzyme activity of recombinant bacterium, the results are shown in Table 1.Algin of the present invention is split
Enzyme Aly-Cob is solved compared with the AlgC-PL7 of document report, expressions of both system is identical, and (expression plasmid is pET-28a (+), table
It is e. coli bl21 (DE3) up to host), under 37 DEG C of condition of culture, Aly-Cob enzyme activities are up to 462U/mL, about AlgC-
15 times of PL7 enzyme activity (~30U/mL).
Expressions of the 1 alginate lyase gene aly-cob of table on different hosts and plasmid
Embodiment 3：Recombinate the zymologic property research of algin catenase Aly-Cob
Recombinant bacterium E.coli BL21pLysS/pET-28a (+)-aly-cob is subjected to induction producing enzyme, and fermentation is obtained
Crude enzyme liquid carry out zymologic property research after purification.
1) optimum temperature and temperature stability of recombination algin catenase Aly-Cob：By the recombination brown alga of debita spissitudo
Glue lyases Aly-Cob solution measures enzyme activity at 25~65 DEG C respectively, by Aly-Cob enzyme solutions respectively in above-mentioned different temperatures
In respectively keep the temperature 30min, measure remaining enzyme activity, highest enzyme activity be defined as 100%.In addition, Aly-Cob enzyme solutions are distinguished
It is kept the temperature in 35,40,45,55,65 DEG C of water-baths.Sample residual enzyme activity is measured by sampling per 10min, determines the enzyme in different temperatures
Under stability.It is final to determine that the optimum temperature of recombination algin catenase Aly-Cob is 45 DEG C, partly declining when less than 55 DEG C
Phase is all higher than 1.0h, at 65 DEG C keep the temperature 1.0h after still have 48.76% opposite enzyme activity, illustrate the enzyme when less than 55 DEG C heat surely
It is qualitative preferable.
2)Na+And K+On recombinating the active influences of Aly-Cob：Due to Na+And K+There is apparent facilitation to Aly-Cob,
Therefore under different NaCl and KCl concentration (0.2,0.4,0.6,0.8,1.0,1.5,2.0,2.5,3.0,4.0molL-1) in
4 DEG C of incubation 1.0h, measure residual enzyme activity, determine best Na at the standard conditions+And K+Concentration and to enzyme generate inhibiting effect
When NaCl and KCl concentration.Any metal ion will be not added with as blank control group, set its corresponding enzyme activity as 100%.
The results show that 1.5molL-1NaCl and 1molL-1KCl it is maximum to the facilitation of recombinase, in 4molL- 1NaCl and 3molL-1Still recombinase unrestraint is acted under the salinity of KCl.The Vibrio sp.YKW-34 of document report and
The resistance to NaCl concentration of algin catenase highest secreted by the Vibrio sp.QY105 and thermophilic salt termite bacterium WX of bacterial strain is respectively 0.1,
0.6 and 0.68molL-1, comparative illustration Aly-Cob is with higher salt tolerance.
3) substrate specificity of Aly-Cob：To study degradation capability of the enzyme to different substrates, the substrate for analyzing the enzyme is special
The opposite sex, the substrate mainly chosen have sodium alginate, PM, PG.Concrete outcome is shown in Table 2, this is the result shows that the enzyme has degradation brown alga
The ability of polysaccharide can not only degrade PG, but also the PM that can degrade for the algal polysaccharide enzyme, therefore, it is determined that the enzyme is a kind of difunctional
Enzyme.
Degradation situation of 2 recombinase of table to different substrates
According to fig. 3,4 as can be seen that enzymolysis 110min after recombinase catabolite have two, three, four, five, six sugar, do not examine
Monosaccharide generation is measured, this catabolite from the recombinase AlgC-PL7 of document report in 110min is different, it is seen that although the two
Similarity is very high in sequence, but there are significant differences on zymologic property.
Embodiment 4：Recombinate applications of the algin catenase Aly-Cob in kelp liquid viscosity reduction
Kelp liquid 3000r/min is centrifuged into 5min, takes supernatant, enzyme solution is added at 40 DEG C, weight is added in 50mL kelp liquid
The group algin catenase total enzyme amount 2000U of Aly-Cob, enzymolysis are constant to reduction sugar amount；Alpha-amylase 2000U is added, is digested
2h is constant to reduction sugar amount.It is sprayed on plant stem-leaf surface by being fitted into spray bottle after enzymolysis liquid centrifugation 10min, there is sterilization and promote
Into the effect of plant-root growth.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill
The people of art can do various change and modification, therefore the protection model of the present invention without departing from the spirit and scope of the present invention
Enclosing be subject to what claims were defined.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>A kind of recombination algin catenase and its construction method and application
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 1059
<212> DNA
<213> Unknown
<220>
<223>It screens and obtains from natural environment
<400> 1
atgcgtaaca cccgtgtgca ccaccccttg gtgacagcat ttctcctcgc tgccagtgca 60
gtcgccattt cagcaccggc tctggccaat gacactcccc ccggagagac attcgatctc 120
gatacctgga agctgactct tccgatggat gccgacggca atggcaaggt ggatgaaatc 180
aaggtcgccg atcttcagtc ctatcgtcac tctgactact tctatctcga cgatgattcc 240
catatggtct tcgtgacgcc caacaaggca ttcacgacac caaattcaag caatgcccgc 300
acggaactgc gccagatgct gcgtggcact gacaccagca tcggcaccca tgatccgaag 360
aacaacttcg cactcgcctc gaatcaacat gccgatgaat tcgctcagat aggcggctac 420
ctttccgcca ccctgcgagt ggaacatgtc gcggagcgtt cgaagaaacc agacaggaag 480
tcagcctact ccgtggtggt tggccagata catgctggca aggaccaggc attgatggag 540
gccgatgaag gtttcgggca tggcaacgag ccactcaaga tcttctacaa gaagctgccg 600
gacgacaaga ctggctccgt gttctggaac tacgagaaga atctcgccaa ggaagatccc 660
aagcgtaccg atgtcagcta cgcggtatgg ggcaacgact ggagcagcaa tgctgatccc 720
ggcaaggaag gtatcgcact gggtgatacc ttcagctaca aggtcgaggt caagggcgac 780
atcatgcatc tgaccttcaa tgccgatggc catccgacac acaatttcga gatcaatctt 840
gccgacaatg tcgatgccaa cggcaaggtg gataacgatg atcttccggc gggctacgcc 900
ggtgactgga tgtacttcaa ggcggggtcc tacaatcagt gcaacaccaa agccagttcc 960
aatgcctgtg aaggtacggg cgtatgggaa accgacaagg ccaacggcga ctacgccaag 1020
gtcgttttca ccaaggttga gagcggtgag atgcaatga 1059
<210> 2
<211> 352
<212> PRT
<213> Unknown
<220>
<223>It screens and obtains from natural environment
<400> 2
Met Arg Asn Thr Arg Val His His Pro Leu Val Thr Ala Phe Leu Leu
1 5 10 15
Ala Ala Ser Ala Val Ala Ile Ser Ala Pro Ala Leu Ala Asn Asp Thr
20 25 30
Pro Pro Gly Glu Thr Phe Asp Leu Asp Thr Trp Lys Leu Thr Leu Pro
35 40 45
Met Asp Ala Asp Gly Asn Gly Lys Val Asp Glu Ile Lys Val Ala Asp
50 55 60
Leu Gln Ser Tyr Arg His Ser Asp Tyr Phe Tyr Leu Asp Asp Asp Ser
65 70 75 80
His Met Val Phe Val Thr Pro Asn Lys Ala Phe Thr Thr Pro Asn Ser
85 90 95
Ser Asn Ala Arg Thr Glu Leu Arg Gln Met Leu Arg Gly Thr Asp Thr
100 105 110
Ser Ile Gly Thr His Asp Pro Lys Asn Asn Phe Ala Leu Ala Ser Asn
115 120 125
Gln His Ala Asp Glu Phe Ala Gln Ile Gly Gly Tyr Leu Ser Ala Thr
130 135 140
Leu Arg Val Glu His Val Ala Glu Arg Ser Lys Lys Pro Asp Arg Lys
145 150 155 160
Ser Ala Tyr Ser Val Val Val Gly Gln Ile His Ala Gly Lys Asp Gln
165 170 175
Ala Leu Met Glu Ala Asp Glu Gly Phe Gly His Gly Asn Glu Pro Leu
180 185 190
Lys Ile Phe Tyr Lys Lys Leu Pro Asp Asp Lys Thr Gly Ser Val Phe
195 200 205
Trp Asn Tyr Glu Lys Asn Leu Ala Lys Glu Asp Pro Lys Arg Thr Asp
210 215 220
Val Ser Tyr Ala Val Trp Gly Asn Asp Trp Ser Ser Asn Ala Asp Pro
225 230 235 240
Gly Lys Glu Gly Ile Ala Leu Gly Asp Thr Phe Ser Tyr Lys Val Glu
245 250 255
Val Lys Gly Asp Ile Met His Leu Thr Phe Asn Ala Asp Gly His Pro
260 265 270
Thr His Asn Phe Glu Ile Asn Leu Ala Asp Asn Val Asp Ala Asn Gly
275 280 285
Lys Val Asp Asn Asp Asp Leu Pro Ala Gly Tyr Ala Gly Asp Trp Met
290 295 300
Tyr Phe Lys Ala Gly Ser Tyr Asn Gln Cys Asn Thr Lys Ala Ser Ser
305 310 315 320
Asn Ala Cys Glu Gly Thr Gly Val Trp Glu Thr Asp Lys Ala Asn Gly
325 330 335
Asp Tyr Ala Lys Val Val Phe Thr Lys Val Glu Ser Gly Glu Met Gln
340 345 350
<210> 3
<211> 27
<212> DNA
<213>Artificial sequence
<400> 3
cgggatccat gcgtaacacc cgtgtgc 27
<210> 4
<211> 29
<212> DNA
<213>Artificial sequence
<400> 4
cccaagcttt cattgcatct caccgctct 29
Claims (10)
1. a kind of gene of coding algin catenase Aly-Cob, which is characterized in that its nucleotide sequence have following feature it
One：
1) there is DNA sequence shown in SEQ ID NO.1；
2) DNA sequence of amino acid sequence shown in coding SEQ ID NO.2；
3) one or several nucleotide of the DNA sequence of SEQ ID NO.1 progress are replaced, missed or added and is obtained
The coding arrived has the active nucleotide sequence of algin catenase.
2. a kind of algin catenase, which is characterized in that have one of following feature：
1) 1-352s or 30-352 amino acids residue sequence of the SEQ ID NO.2 since aminoterminal；
2) one or several amino acid substitutions, deletions, or additions are carried out to amino acid sequence shown in SEQ ID NO.2 and is formed
Have the active amino acid sequence of algin catenase.
3. a kind of method preparing the algin catenase described in claim 2, which is characterized in that include the following steps：
(1) alginate lyase gene is cloned into expression vector, obtains recombinant plasmid；
(2) by recombinant plasmid transformed host strain, the genetic engineering bacterium of production algin catenase is obtained.
4. according to the method described in claim 3, it is characterized in that, expression vector described in step (1), refers to Escherichia coli table
Up to carrier, Yeast expression carrier, hay bacillus expression vector, Corynebacterium glutamicum expression vector, lactic acid bacteria expression vectors, strepto-
Bacterium expression vector, phage vector, filamentous fungi expression vector, plant expression vector, insect expression vector or mammal are thin
Cellular expression carrier.
5. method according to claim 3 or 4, it is characterised in that：The host strain, it includes Escherichia to refer to
Escherichia coli host including coli BL21, Escherichia coli JM109 or Escherichia coli DH5 α is thin
Born of the same parents, the ferment including Saccharomyces cerevisiae, Pichiapas toris or Kluyveromyces lactis
Female bacterium host cell, the hay bacillus place including Bacillus subtilis R25, Bacillus subtilis 9920
Chief cell, the corynebacterium glutamicum including Corynebacterium glutamicum including Lacticacid
Lactic acid bacteria host cell including bacteria COCC101, the actinomyces host including Streptomyces spp. are thin
Born of the same parents including Trichoderma viride, Trichoderma reesei, Aspergillus niger, Aspergillus
The insect including filamentous fungal host cell including Bombyx mori, Antharaea eucalypti including nidulans
Cell, or the food in one's mouth including Chinese hamster ovary cell CHO, baby hamster kidney cell BHK, CHL cells CHL
Newborn zooblast.
6. a kind of genetic engineering bacterium of the algin catenase described in expression claim 2.
7. application of the genetic engineering bacterium described in claim 6 in algin catenase is produced in fermentation.
8. application of the algin catenase in degradation alginate or algin described in claim 2, which is characterized in that extremely
There is one of following purposes less：
1) it is used to be broken the glycosidic bond of alginate or algin, obtains alginate oligosaccharides；
2) for the alginate component in degrade red algae or brown alga cell wall, protoplast is extracted；
Or it 3) is used to destroy pathogenic bacteria pseudomonas aeruginosa (Pseudomonas aeruginosa) and is formed by brown alga in mycoderm
Sour sodium ingredient.
9. application as claimed in claim 8, it is characterised in that：The inscribe algin catenase Aly-Cob and other sources
Algin degrading enzyme or after other kinds of enzyme is used in combination, the height for containing alginate for Synergistic degradation glues raw material.
10. carrying the carrier of gene or genetic engineering bacterium or transgenic cell line described in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810325487.9A CN108285900B (en) | 2018-04-12 | 2018-04-12 | Recombinant alginate lyase and construction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810325487.9A CN108285900B (en) | 2018-04-12 | 2018-04-12 | Recombinant alginate lyase and construction method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108285900A true CN108285900A (en) | 2018-07-17 |
| CN108285900B CN108285900B (en) | 2020-06-09 |
Family
ID=62834304
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810325487.9A Active CN108285900B (en) | 2018-04-12 | 2018-04-12 | Recombinant alginate lyase and construction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108285900B (en) |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108690857A (en) * | 2018-05-22 | 2018-10-23 | 齐鲁工业大学 | A kind of preparation method of high-purity brown alga oligose |
| CN109295043A (en) * | 2018-10-19 | 2019-02-01 | 中国科学院天津工业生物技术研究所 | A kind of novel algin catenase, preparation method and application |
| CN109750022A (en) * | 2019-03-27 | 2019-05-14 | 中科荣信（苏州）生物科技有限公司 | A kind of algin catenase Alg2A and its preparation method and application |
| CN109852601A (en) * | 2019-03-04 | 2019-06-07 | 江南大学 | It is a kind of can efficient application N- glycosylation algin catenase mutant and construction of genetic engineering method |
| CN110257410A (en) * | 2019-07-24 | 2019-09-20 | 江南大学 | A kind of gene encoding algin catenase |
| CN110331137A (en) * | 2019-06-03 | 2019-10-15 | 中国海洋大学 | A kind of algin catenase and preparation method thereof |
| CN110452919A (en) * | 2019-09-10 | 2019-11-15 | 南京工业大学 | Truncated alginate lyase Aly7B-CDII gene and application thereof |
| CN110656054A (en) * | 2019-11-08 | 2020-01-07 | 杭州师范大学 | Recombinant trichoderma reesei for extracellularly secreting alginate lyase and application thereof |
| CN110885850A (en) * | 2019-12-06 | 2020-03-17 | 五洲丰农业科技有限公司 | Method for preparing alginate oligosaccharides and method for improving quality of turbot by using alginate oligosaccharides |
| CN111269907A (en) * | 2020-04-03 | 2020-06-12 | 江南大学 | Alginate lyase mutant based on loop region transformation and application thereof |
| CN111424027A (en) * | 2020-03-31 | 2020-07-17 | 江南大学 | Site-directed mutagenesis modified alginate lyase mutant and application thereof |
| CN112143724A (en) * | 2019-06-28 | 2020-12-29 | 中国科学院青岛生物能源与过程研究所 | Acetyl sodium alginate esterase and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695341A (en) * | 2013-12-16 | 2014-04-02 | 江南大学 | Alginate lyase secreted from marine bacterium and preparation method thereof |
| CN105821063A (en) * | 2015-01-05 | 2016-08-03 | 中国科学院大连化学物理研究所 | Incision alginate lyase Alg2B and coding gene, preparation and application thereof |
| CN109852601A (en) * | 2019-03-04 | 2019-06-07 | 江南大学 | It is a kind of can efficient application N- glycosylation algin catenase mutant and construction of genetic engineering method |
-
2018
- 2018-04-12 CN CN201810325487.9A patent/CN108285900B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695341A (en) * | 2013-12-16 | 2014-04-02 | 江南大学 | Alginate lyase secreted from marine bacterium and preparation method thereof |
| CN105821063A (en) * | 2015-01-05 | 2016-08-03 | 中国科学院大连化学物理研究所 | Incision alginate lyase Alg2B and coding gene, preparation and application thereof |
| CN109852601A (en) * | 2019-03-04 | 2019-06-07 | 江南大学 | It is a kind of can efficient application N- glycosylation algin catenase mutant and construction of genetic engineering method |
Non-Patent Citations (4)
| Title |
|---|
| GENBANK: "CP017114.1", 《NCBI》 * |
| HISASHI YAGI等: "Purification and characterization of a novel alginate lyase from the marine bacterium Cobetia sp. NAP1 isolated from brown algae", 《BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY》 * |
| JIN-SONG GONG 等: "Purification and characterization of a high salt-tolerant alginate lyase from Cobetia sp.WG-007", 《BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY》 * |
| 刘旭梅等: "产褐藻胶裂解酶菌株 Cobetia sp. WG-007 的筛选及发酵优化", 《食品与生物技术学报》 * |
Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108690857A (en) * | 2018-05-22 | 2018-10-23 | 齐鲁工业大学 | A kind of preparation method of high-purity brown alga oligose |
| CN109295043A (en) * | 2018-10-19 | 2019-02-01 | 中国科学院天津工业生物技术研究所 | A kind of novel algin catenase, preparation method and application |
| CN109295043B (en) * | 2018-10-19 | 2021-02-05 | 中国科学院天津工业生物技术研究所 | Alginate lyase, and preparation method and application thereof |
| CN109852601A (en) * | 2019-03-04 | 2019-06-07 | 江南大学 | It is a kind of can efficient application N- glycosylation algin catenase mutant and construction of genetic engineering method |
| CN109852601B (en) * | 2019-03-04 | 2023-04-07 | 江南大学 | N-glycosylation alginate lyase mutant capable of being efficiently applied and construction method of genetic engineering bacteria |
| CN109750022A (en) * | 2019-03-27 | 2019-05-14 | 中科荣信（苏州）生物科技有限公司 | A kind of algin catenase Alg2A and its preparation method and application |
| CN110331137A (en) * | 2019-06-03 | 2019-10-15 | 中国海洋大学 | A kind of algin catenase and preparation method thereof |
| CN112143724A (en) * | 2019-06-28 | 2020-12-29 | 中国科学院青岛生物能源与过程研究所 | Acetyl sodium alginate esterase and application thereof |
| CN112143724B (en) * | 2019-06-28 | 2022-07-26 | 中国科学院青岛生物能源与过程研究所 | Acetyl sodium alginate esterase and application thereof |
| CN110257410A (en) * | 2019-07-24 | 2019-09-20 | 江南大学 | A kind of gene encoding algin catenase |
| CN110452919A (en) * | 2019-09-10 | 2019-11-15 | 南京工业大学 | Truncated alginate lyase Aly7B-CDII gene and application thereof |
| CN110656054A (en) * | 2019-11-08 | 2020-01-07 | 杭州师范大学 | Recombinant trichoderma reesei for extracellularly secreting alginate lyase and application thereof |
| CN110885850A (en) * | 2019-12-06 | 2020-03-17 | 五洲丰农业科技有限公司 | Method for preparing alginate oligosaccharides and method for improving quality of turbot by using alginate oligosaccharides |
| CN111424027A (en) * | 2020-03-31 | 2020-07-17 | 江南大学 | Site-directed mutagenesis modified alginate lyase mutant and application thereof |
| CN111424027B (en) * | 2020-03-31 | 2022-03-01 | 江南大学 | Site-directed mutagenesis modified alginate lyase mutant and application thereof |
| CN111269907A (en) * | 2020-04-03 | 2020-06-12 | 江南大学 | Alginate lyase mutant based on loop region transformation and application thereof |
| CN111269907B (en) * | 2020-04-03 | 2022-03-01 | 江南大学 | Alginate lyase mutant based on loop region transformation and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108285900B (en) | 2020-06-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108285900A (en) | A kind of recombination algin catenase and its construction method and application | |
| CN106350531A (en) | Alginate lyase gene and application thereof | |
| WO2017197546A1 (en) | Β-mannanase mrmman5a and encoding gene and application thereof | |
| CN105713889A (en) | Alginate lyase Alga and coding gene and application thereof | |
| CN112941052B (en) | Chitosanase OUC-T613 and application thereof | |
| CN113862241A (en) | Chitosanase Csncv, mutant CsnB thereof and application of mutant CsnB | |
| CN111893126A (en) | Alkaline protease gene, alkaline protease, preparation method and application thereof | |
| CN112111472B (en) | Novel beta-xylosidase and preparation thereof | |
| CN108102936B (en) | Kluyveromyces lactis mutant strain, glycosidase thereof and application thereof | |
| CN111996205A (en) | Chitinase gene, chitinase and preparation method and application thereof | |
| CN110511917A (en) | A kind of deacetylase and its encoding gene and application | |
| CN110643622A (en) | Alginate lyase gene and application thereof | |
| CN112195168B (en) | Thermophilic chitinase Chi304 mutant and preparation method and application thereof | |
| CN103525782A (en) | Organophosphorus pesticide degrading enzyme gene and application thereof | |
| CN101372693A (en) | Heat resisting cellulase gene, recombinant engineering bacterium, heat resisting cellulase and use | |
| CN112574980B (en) | Recombinant alginate lyase with thermal stability and high enzyme activity and application thereof | |
| CN104877979B (en) | A kind of its encoding gene of the β mannonases of first genomic source and its expression | |
| CN106754987A (en) | A kind of polysaccharide cracks monooxygenase LPMO M1 encoding genes and its enzyme and preparation method and application | |
| CN110093326B (en) | Extracellular AA9 family polysaccharide monooxygenase EpLPMOa and application thereof | |
| CN105154417B (en) | The acidic cellulase and its gene of a kind of originated from fungus and application | |
| CN107603967A (en) | A kind of chitosan enzyme CSN4 and its encoding gene and application | |
| KR20100040438A (en) | A novel agarase and an enzymatic production method of agarooligosaccharide from agarose using the same | |
| CN101906405A (en) | Cloning of inulin ftructotransferase and efficient expression thereof | |
| CN110484525A (en) | A kind of heat-resisting N-acetylglucosamine deacetylase and its encoding gene and application | |
| CN112725315B (en) | Application of chitosanase and mutant thereof in preparation of chitosan oligosaccharide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20220425 Address after: 214000 1800 Lihu Avenue, Binhu District, Wuxi, Jiangsu Patentee after: Jiangnan University Patentee after: Chengdu Yongan Yuanhe Biotechnology Co., Ltd Address before: No. 1800 road 214122 Jiangsu Lihu Binhu District City of Wuxi Province Patentee before: Jiangnan University |